Cardiovascular activity of Micrurus lemniscatus lemniscatus (South American coralsnake) venom.

Envenomation by coralsnakes (Micrurus spp.) is characterized by blockade of peripheral neurotransmission mediated by the presence of α- and ß-neurotoxins. However, little is known about their cardiovascular activity. Micrurus lemniscatus lemniscatus is a coralsnake found in the Amazon basin and occasionally causes envenomation in humans. In this study, we examined the hemodynamic, vascular and atrial responses to M. l. lemniscatus venom. Anesthetized rats were used for hemodynamic and electrocardiogram (ECG) recordings; in vitro experiments were carried out in rat isolated thoracic aorta and atria preparations. In vivo, venom (0.1 and 0.3 mg/kg) caused immediate and persistent hypotension that was maximal within the first minute with both doses being lethal after ~40 and ~20 min, respectively. ECG, heart and respiratory rates were not altered during the transient hypotension phase induced by venom but all altered prior to death. There was no evidence of myonecrosis in cardiac muscle tissue, pulmonary hemorrhage nor thrombosis in anesthetized rats exposed to venom. In vitro, venom (10 µg/ml) did not contract aortic strips nor affected the maximal responses to pre-contraction with phenylephrine (PE, 0.0001-30 µM) in strips with and without endothelium. However, venom (10 µg/ml) relaxed aortic strips with endothelium pre-contracted with PE. In aortic strips pre-contracted with PE, venom prevented acetylcholine (0.0001-30 µM)-induced relaxation in strips with endothelium without affecting relaxation induced by sodium nitroprusside (0.1-100 nM) in strips without endothelium. Venom (30 µg/ml) produced a transient increase of atrial contractile force without affecting atrial rate. Taken together these findings indicate a predominantly vascular action of the venom, most likely involving toxins interacting with muscarinic receptors.

Mirabegron elicits rat corpus cavernosum relaxation and increases in vivo erectile response.

Mirabegron is the first ß3-adrenoceptor agonist approved on the market and may offer beneficial pharmacological action in patients with overactive bladder and erectile dysfunction. Here, we further investigate the mechanisms by which mirabegron induces rat corpus cavernosum (CC) relaxation. Adult male Wistar rats were used. The CC were isolated for in vitro functional assays and ß-adrenoceptors subtypes mRNA expression evaluation. Animals were treated orally with mirabegron (30 mg/kg, 3 h), tadalafil (10 mg/kg, 3 h) or both for intracavernous pressure (ICP). Intracellular levels of cAMP and cGMP were also determined. The ß1-, ß2- and ß3-adrenoceptors subtypes were expressed in rat CC. Mirabegron produced concentration-dependent CC relaxations that were unaffected by the ß1-, ß2- or ß3-adrenoceptor antagonists atenolol (1 µM), ICI-118,551 (1 µM) and L748,337 (10 µM), respectively. Mirabegron-induced relaxations were not affected by the phosphodiesterase type 4 inhibitor, rolipram, or the adenylyl cyclase selective inhibitor, SQ 22,536. Potassium channel- or calcium influx-blockade are not involved in mirabegron-induced relaxations. In contrast, mirabegron produced rightward shifts in the contractile response induced by the α1-adrenoceptor agonist, phenylephrine. Finally, cavernous nerve stimulation caused frequency-dependent ICP increases, which were significantly increased in rats treated with mirabegron in a similar degree of tadalafil-treated rat, without promoting a significant cAMP or cGMP accumulation. Together, our results demonstrate that mirabegron induced CC relaxation through α1-adrenoceptor blockade. Care should be taken to translate the effect of mirabegron into the clinic, especially when using rat as an animal model of erectile dysfunction.

Development, Validation of LC-MS/MS Method and Determination of Pharmacokinetic Parameters of the Stroke Neuroprotectant Neurounina-1 in Beagle Dog Plasma After Intravenous Administration.

Neurounina-1 [chemical name: 7-nitro-5-phenyl-1-(pyrrolidin-1-ylmethyl)-1H-benzo[e][1,4]diazepin-2(3H)-one] is a new compound provided with relevant neuroprotective effect during stroke and in neonatal hypoxia by increasing the Na+/Ca2+ exchanger (NCX) isoforms NCX1 and NCX2 activity. This study shows for the first time, the development and validation of a sensitive and selective method for analysis of neurounina-1 in beagle dog plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The sample preparation consisted of extraction of the analyte and the internal standard (IS) (ropivacaine) from plasma (50 µL) by liquid-liquid extraction using acetonitrile (100 µL). The selected reaction monitoring mode of the positive ion was performed and the precursor to the product ion transitions of m/z 365 > 83 and m/z 275 > 126 were used to measure the derivative of neurounina-1 and ropivacaine. The chromatographic separation was achieved using a Phenomenex C18 Luna (150 mm × 4.6 mm × 5 µm) analytical column with an isocratic mobile phase composed of methanol/acetonitrile/water (50/40/10, v/v/v) + 0.1% formic acid + 1 M ammonium formate. The method was linear over a concentration range of 1-500 ng/mL. The method was applied to evaluate the pharmacokinetics of neurounina-1 after a single intravenous administration of three different doses (0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg) to beagle dogs (n = 5). The mean AUC0-tlast values were 26.10, 115.81, and 257.28 ng∗h/mL following intravenous administration of 0.1, 0.3, and 1 mg/kg, respectively. Linear pharmacokinetics was observed up to 1.0 mg/kg. The neurounina-1 was rapidly eliminated, with mean CL values of 46.24, 47.57, and 69.15 L/h, Vd of 130.31, 154.15, and 210.79 L and t1/2 of 2.14, 2.54, and 2.04 h after intravenous administration of 0.1, 0.3, and 1 mg/kg, respectively. This new analytical method allows the rapid determination of the neurounina-1, a new developed compound, able to exert a remarkable neuroprotective effect in the low nanomolar range.

Pharmacological and transcriptomic characterization of the nitric oxide pathway in aortic rings isolated from the tortoise Chelonoidis carbonaria.

In this study the nitric oxide (NO)-soluble guanylate cyclase (sGC) and phosphodiesterase-5 (PDE-5) pathway was characterized in tortoise Chelonoidis carbonaria aorta. Concentration response curves (CCR) to ATP, ADP, AMP, adenosine and histamine were performed in the presence and absence of L-NAME in aorta pre-contracted with ACh (3 µM). CCR to SNP, BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator) and tadalafil (PDE-5 inhibitor) were constructed in the presence and absence of ODQ (10 µM). ATP (pEC50 6.1 ±â€¯0.1), ADP (pEC50 6.0 ±â€¯0.2), AMP (pEC50 6.8 ±â€¯0.1) and histamine (pEC50 6.8 ±â€¯0.12) relaxed Chelonoidis aorta and the addition of L-NAME reduced their efficacy (p < .05). Adenosine effects (pEC50 6.6 ±â€¯0.1) were not changed in the presence of L-NAME. SNP (pEC50 7.5 ±â€¯0.7; Emax 102.2 ±â€¯2.5%), BAY 41-2272 (pEC50 7.3 ±â€¯0.2; Emax 130.3 ±â€¯10.2%), BAY 60-2770 (pEC50 11.4 ±â€¯0.1; Emax 130.3 ±â€¯6.5%) and tadalafil (pEC50 6.7 ±â€¯0.3; Emax 121.3 ±â€¯15.3%) relaxed Chelonoidis aorta. The addition of ODQ reduced the SNP and tadalafil maximum response (p < .05) and promoted 63 fold right shift on BAY 41-2272 curve. In contrast, no alteration was observed on BAY 60-2770 response. Transcriptomic analysis for eNOS and sGC were found in aorta and brain libraries with high homology when compared with human transcripts. The NO-sGC-PDE-5 is functionally present in Chelonoidis aorta with a functional and genomic similarity to mammalian vessels. Unlike most of mammalian vessels, ACh did not cause endothelium-dependent relaxation in Chelonoidis carbonaria aortic rings.

Pharmacokinetics of Riluzole in Beagle Dogs.

BACKGROUND: Riluzole is a benzothiazole anticonvulsant used in the treatment of patients with amyotrophic lateral sclerosis and it is being investigated for clinical use in patients with spinal cord injury. The present study evaluated the pharmacokinetics of riluzole in beagle dogs after oral dose administration. METHODS: The oral doses (1.5, 5, 15 and 50 mg/kg) of riluzole were administered to beagle dogs and blood samples were collected from 0 h to 24 h post drug administration. Riluzole was quantified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RESULTS: The method was sensitive, precise, accurate and selective to riluzole quantification in plasma of beagle dogs. The pharmacokinetics following oral administration was linear from 1.5 to 15 mg/kg and the t1/2 was 2.16, 1.5, 1.8 and 3.0 h after oral administration of 1.5, 5.0, 15 and 50 mg/kg riluzole. CONCLUSION: The riluzole pharmacokinetics was linear up to 15 mg/kg and had a significantlyshorter t1/2 in beagle dogs than in humans.

The rabbit vagina as an in vivo model for vaginal fenticonazole permeability and toxicity.

INTRODUCTION: Vaginal route is often used in topical antifungal formulations. Vaginal permeability assays are generally performed as in vitro tests. METHOD: An in vivo vaginal permeability assay was developed using female rabbits. Fenticonazole permeability was evaluated by assessing fenticonazole bioavailability in plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS). Toxicity was monitored histopathologically after 8 consecutive days of antifungal treatment (20 mg/animal). RESULTS: The method of quantification was linear with a lower limit of quantification (LLOQ) of (0.1 ng/mL). The area-under-the-curves (AUC) of fenticonazole on day 1 and 8 of treatment were 280.3 ±â€¯86.1 ng/mL ∗ h and 805.7 ±â€¯252.4 ng/mL ∗ h, respectively. The calculated systemic bioavailability was 12.73% ±â€¯0.14%. No signs of toxicity were observed both macroscopically and histologically after 8 days fenticonazole treatment. DISCUSSION: The plasma levels of fenticonazole observed in rabbits are similar to that observed in human. Rabbit vagina may be a suitable model to evaluate vaginal antifungal formulations.

Tetrodotoxin-insensitive electrical field stimulation-induced contractions on Crotalus durissus terrificus corpus cavernosum.

Reptiles are the first amniotes to develop an intromitent penis, however until now the mechanisms involved in the electrical field stimulation-induced contraction on corpora cavernosa isolated from Crotalus durissus terrificus were not investigated. Crotalus and rabbit corpora cavernosa were mounted in 10 mL organ baths for isometric tension recording. Electrical field stimulation (EFS)-induced contractions were performed in presence/absence of phentolamine (10 µM), guanethidine (30 µM), tetrodotoxin (1 µM and 1mM), A-803467 (10 µM), 3-iodo-L-Tyrosine (1 mM), salsolinol (3 µM) and a modified Krebs solution (equimolar substitution of NaCl by N-methyl-D-glucamine). Immuno-histochemistry for tyrosine hydroxylase was also performed. Electrical field stimulation (EFS; 8 Hz and 16 Hz) caused contractions in both Crotalus and rabbit corpora cavernosa. The contractions were abolished by previous incubation with either phentolamine or guanethidine. Tetrodotoxin (1 µM) also abolished the EFS-induced contractions of rabbit CC, but did not affect EFS-induced contractions of Crotalus CC. Addition of A-803467 (10 µM) did not change the EFS-induced contractions of Crotalus CC but abolished rabbit CC contractions. 3-iodo-L-Tyrosine and salsolinol had no effect on EFS-induced contractions of Crotalus CC and Rabbit CC. Replacement of NaCl by N- Methyl-D-glucamine (NMDG) abolished EFS-induced contractions of rabbit CC, but did not affect Crotalus CC. The presence of tyrosine hydroxylase was identified in endothelial cells only of Crotalus CC. Since the EFS-induced contractions of Crotalus CC is dependent on catecholamine release, insensitive to TTX, insensitive to A803467 and to NaCl replacement, it indicates that the source of cathecolamine is unlikely to be from adrenergic terminals. The finding that tyrosine hydroxylase is present in endothelial cells suggests that these cells can modulate Crotalus CC tone.

Micturition dysfunction in four-month old ovariectomized rats: Effects of testosterone replacement.

AIMS: Androgen deficiency has been implicated in urological complications of postmenopausal women. This study examined the effects of testosterone replacements on the lower urinary tract dysfunction in 4-month old ovariectomized (OVX) rats. MAIN METHODS: Sprague-Dawley female rats were OVX bilaterally. Three months later, rats received single intramuscular injections of testosterone undecanoate. Cystometric study, and bladder and urethra smooth muscle reactivities were evaluated. KEY FINDINGS: Ovariectomy reduced by 65% (p<0.05) the serum testosterone levels. Testosterone replacement at 5mg/kg restored serum hormone levels to baseline, whereas 10mg/kg produced 14-fold higher testosterone levels. OVX rats exhibited significant increases of body weight, perigonadal fat and blood pressure, and reduced uterus weight, but none of these parameters were changed by testosterone replacements. OVX rats exhibited micturition dysfunction characterized by increases of basal pressure, threshold pressure, voiding frequency and post-voiding pressure. In addition, the bladder contractions induced by electrical-field stimulation (EFS) and carbachol were significantly reduced, whereas angiotensin II-induced urethral contractions were significantly increased in OVX rats. Testosterone replacement at 10mg/kg (but not at 5mg/kg) dose fully normalized the in vivo micturition dysfunction, as well as the in vitro bladder and urethral alterations. Testosterone (10mg/kg) also significantly potentiated the bladder relaxations induced by the ß3-adrenoceptor agonist mirabegron. The protective effects of testosterone were not modified by concomitant treatment with the aromatase inhibitor letrozole (2.5mg/kg, 4weeks). SIGNIFICANCE: The improvement of micturition dysfunction by testosterone replacement suggests that androgen therapy might be of therapeutic benefit for urological complications associated with post-menopause.

Effects of nitric oxide inhibitors in mice with bladder outlet obstruction

ABSTRACT Purpose To investigate the lower urinary tract changes in mice treated with L-NAME, a non-selective competitive inhibitor of nitric oxide synthase (NOS), or aminoguanidine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), after 5 weeks of partial bladder outlet obstruction (BOO), in order to evaluate the role of constitutive and non-constitutive NOS in the pathogenesis of this experimental condition. Materials and Methods C57BL6 male mice were partially obstructed and randomly allocated into 6 groups: Sham, Sham + L-NAME, Sham + aminoguanidine, BOO, BOO + L-NAME and BOO + aminoguanidine. After 5 weeks, bladder weight was obtained and cystometry and tissue bath contractile studies were performed. Results BOO animals showed increase of non-voiding contractions (NVC) and bladder capacity, and also less contractile response to Carbachol and Electric Field Stimulation. Inhibition of NOS isoforms improved bladder capacity and compliance in BOO animals. L-NAME caused more NVC, prevented bladder weight gain and leaded to augmented contractile responses at muscarinic and electric stimulation. Aminoguanidine diminished NVC, but did not avoid bladder weight gain in BOO animals and did not improve contractile responses. Conclusion It can be hypothesized that chronic inhibition of three NOS isoforms in BOO animals leaded to worsening of bladder function, while selective inhibition of iNOS did not improve responses, what suggests that, in BOO animals, alterations are related to constitutive NOS.

Effects of nitric oxide inhibitors in mice with bladder outlet obstruction.

PURPOSE: To investigate the lower urinary tract changes in mice treated with L-NAME, a non-selective competitive inhibitor of nitric oxide synthase (NOS), or aminoguanidine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), after 5 weeks of partial bladder outlet obstruction (BOO), in order to evaluate the role of constitutive and non-constitutive NOS in the pathogenesis of this experimental condition. MATERIALS AND METHODS: C57BL6 male mice were partially obstructed and randomly allocated into 6 groups: Sham, Sham + L-NAME, Sham + aminoguanidine, BOO, BOO + L-NAME and BOO + aminoguanidine. After 5 weeks, bladder weight was obtained and cystometry and tissue bath contractile studies were performed. RESULTS: BOO animals showed increase of non-voiding contractions (NVC) and bladder capacity, and also less contractile response to Carbachol and Electric Field Stimulation. Inhibition of NOS isoforms improved bladder capacity and compliance in BOO animals. L-NAME caused more NVC, prevented bladder weight gain and leaded to augmented contractile responses at muscarinic and electric stimulation. Aminoguanidine diminished NVC, but did not avoid bladder weight gain in BOO animals and did not improve contractile responses. CONCLUSION: It can be hypothesized that chronic inhibition of three NOS isoforms in BOO animals leaded to worsening of bladder function, while selective inhibition of iNOS did not improve responses, what suggests that, in BOO animals, alterations are related to constitutive NOS.

The soluble guanylyl cyclase activator BAY 60-2770 potently relaxes the pulmonary artery on congenital diaphragmatic hernia rabbit model.

PURPOSE: Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypertension which is often difficult to manage, and a significant cause of morbidity and mortality. In this study, we have used a rabbit model of CDH to evaluate the effects of BAY 60-2770 on the in vitro reactivity of left pulmonary artery. METHODS: CDH was performed in New Zealand rabbit fetuses (n = 10 per group) and compared to controls. Measurements of body, total and left lung weights (BW, TLW, LLW) were done. Pulmonary artery rings were pre-contracted with phenylephrine (10 µM), after which cumulative concentration-response curves to glyceryl trinitrate (GTN; NO donor), tadalafil (PDE5 inhibitor) and BAY 60-2770 (sGC activator) were obtained as well as the levels of NO (NO3/NO2). RESULTS: LLW, TLW and LBR were decreased in CDH (p < 0.05). In left pulmonary artery, the potency (pEC50) for GTN was markedly lower in CDH (8.25 ± 0.02 versus 9.27 ± 0.03; p < 0.01). In contrast, the potency for BAY 60-2770 was markedly greater in CDH (11.7 ± 0.03 versus 10.5 ± 0.06; p < 0.01). The NO2/NO3 levels were 62 % higher in CDH (p < 0.05). CONCLUSION: BAY 60-2770 exhibits a greater potency to relax the pulmonary artery in CDH, indicating a potential use for pulmonary hypertension in this disease.

L-Carnitine supplementation impairs endothelium-dependent relaxation in mesenteric arteries from rats.

L-Carnitine (L-Car) is taken as fat burner. The risks of L-Car supplementation for the cardiovascular system are unclear. We evaluated the relaxing responses of the mesenteric and aorta rings from rats after four weeks of L-Car supplementation and/or physical training. Concentration response curves to acetylcholine (ACh) and sodium nitroprusside (SNP), as well as cyclic GMP levels, superoxide dismutase (SOD) activity and malondialdehyde (MDA) were evaluated. Physical training decreased body weight gain that was potentiated by L-Car. In mesenteric rings, L-Car impaired endothelium-dependent relaxation whereas endothelium independent relaxation was increased. In aorta, exercise improved endothelium-dependent relaxation; however, it was partially inhibited by L-Car. SNP-induced relaxation was similar in aorta of all groups. Basal cGMP were increased in aorta of exercised rats. SOD activity and MDA levels were unaltered. In conclusion, L-Car and physical exercise promotes body weight loss; however, it impairs endothelium-dependent vaso-relaxation possibly involving alterations in muscarinic receptors/eNOS/NO signalling pathway in mesenteric artery.

The Evolutionary Implications of Hemipenial Morphology of Rattlesnake Crotalus durissus terrificus (Laurent, 1768) (Serpentes: Viperidae: Crotalinae).

Most amniotes vertebrates have an intromittent organ to deliver semen. The reptile Sphenodon and most birds lost the ancestral penis and developed a cloaca-cloaca mating. Known as hemipenises, the copulatory organ of Squamata shows unique features between the amniotes intromittent organ. They are the only paired intromittent organs across amniotes and are fully inverted and encapsulated in the tail when not in use. The histology and ultrastructure of the hemipenes of Crotalus durissus rattlesnake is described as the evolutionary implications of the main features discussed. The organization of hemipenis of Crotalus durissus terrificus in two concentric corpora cavernosa is similar to other Squamata but differ markedly from the organization of the penis found in crocodilians, testudinata, birds and mammals. Based on the available data, the penis of the ancestral amniotes was made of connective tissue and the incorporation of smooth muscle in the framework of the sinusoids occurred independently in mammals and Crotalus durissus. The propulsor action of the muscle retractor penis basalis was confirmed and therefore the named should be changed to musculus hemipenis propulsor.The retractor penis magnus found in Squamata has no homology to the retractor penis of mammals, although both are responsible for the retraction of the copulatory organ.

Increased contractility and impaired relaxation of the left pulmonary artery in a rabbit model of congenital diaphragmatic hernia.

PURPOSE: Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypertension which is often difficult to manage and a significant cause of morbidity and mortality. Our aim was to study the pulmonary artery reactivity in an animal model of CDH. METHODS: To investigate the reactivity of the aorta and left pulmonary artery in a rabbit model of CDH, we studied the in vitro responses to the α1-adrenoceptor agonist phenylephrine (PE) and to both the muscarinic receptor agonist (ACh) and the nitric oxide (NO) donor sodium nitroprusside (SNP). Rabbits underwent surgery at 25 days of gestation. CDH was created in one fetus per horn (n = 8). Remaining fetuses were considered controls (n = 18). At term (30 days), the lung, left pulmonary artery, and aorta were dissected. In a separate group, endothelium was mechanically removed. RESULTS: There were no differences in the contractile and relaxing responses of aorta in all groups. In left pulmonary artery, PE-induced contractions were significantly greater (p < 0.05) in CDH when compared with control group. The increased responsiveness to PE in CDH group was similar to that found in pulmonary artery without endothelium. The ACh-induced pulmonary artery relaxation was markedly reduced in CDH when compared with control group (p < 0.05), whereas no differences were found for SNP. CONCLUSION: Our results show increased contractility and impairment in endothelium-dependent relaxation of pulmonary artery in CDH, mimicking an endothelial dysfunction, with preserved response to endothelium-independent mechanism.

Vas deferens smooth muscle responses to the nitric oxide-independent soluble guanylate cyclase stimulator BAY 41-2272.

The nitric oxide-cGMP signaling pathway modulates the ejaculatory functions. The nitric oxide (NO)-independent soluble guanylate cyclase haem-dependent stimulator BAY 41-2272 potently relaxes different types of smooth muscles. However, no study investigated its effects in vas deferens smooth muscle. Therefore, we designed experiments to evaluate the in vitro relaxing responses of vas deferens to BAY 41-2272. The effects of prolonged oral intake with BAY 41-2272 in vas deferens contractions of rats treated chronically with the NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME) were also investigated. BAY 41-2272 (0.001-100 µM) produced concentration-dependent relaxations in the prostatic and epididymal portions of vas deferens, an effect markedly reduced by the soluble guanylate cyclase inhibitor ODQ (100 µM). BAY 41-2272 significantly increased cGMP levels that were fully prevented by ODQ. In separate protocols, rats received L-NAME (20mg/rat/day) concomitantly with BAY 41-2272 (10mg/kg/day, 4 weeks), after which vas deferens contractions to electrical-field stimulation and noradrenaline were achieved. Electrical-field stimulation (1-32 Hz) evoked frequency-dependent contractions that were significantly enhanced in L-NAME-treated rats. Co-treatment with BAY 41-2272 fully reversed the increased contractile responses in L-NAME group. Noradrenaline (0.01-100 µM)-induced contractions were also greater in L-NAME-treated rats, and that was normalized by BAY 41-2272. In conclusion, BAY 41-2272 potently relaxes in vitro rat vas deferens smooth muscle and elevates the cGMP levels in an ODQ-sensitive manner. Moreover, prolonged oral intake with BAY 41-2272 restores the enhanced contractile vas deferens activity in rats treated with L-NAME. NO-independent soluble guanylate cyclase stimulators may be an alternative treatment for premature ejaculation.

Immunohistochemical and functional characterization of nitric oxide signaling pathway in isolated aorta from Crotalus durissus terrificus.

We characterized the nitric oxide (NO)-cyclic GMP-phosphodiesterase-5 (PDE5) pathway in Crotalus durissus terrificus aorta. Concentration responses curves to acetylcholine (ACh), sodium nitroprusside (SNP), BAY41-2272 (soluble guanylyl cyclase [sGC] stimulator), BAY60-2770 (sGC activator) and tadalafil (PDE5 inhibitor) were constructed in phenylephrine (10 µM)-precontracted tissues with intact (E(+)) or denuded (E(-)) endothelium. ACh (0.0001-10 µM) and SNP (0.0001-10 µM) relaxed aorta, which were reduced by the NO synthase (L-NAME,100 µM) or the sGC inhibitors (ODQ, 10 µM). Tadalafil (0.0001-10 µM) relaxed E(+) rings with potency (pEC(50)) and maximal response (E(max)) values of 7.34±0.02 and 105±8%, respectively. E(-) or ODQ treatment significantly (P<0.05) reduced tadalafil relaxations (66±18% and 71±7%, respectively). BAY41-2272 (0.0001-300 nM) produced concentration-dependent relaxations in E(+) rings, which were reduced by addition of either ODQ or L-NAME (16.0- and 5.2-fold rightward shifts, respectively). The relaxation of BAY60-2770 was markedly potentiated by ODQ and L-NAME (41.0- and 9.7-fold leftward shifts, respectively), whereas in E(-) the pEC(50) values were shifted by 7-fold to the right. Immunohistochemistry, followed validation by transcriptomic analysis, revealed the presence of eNOS in endothelium, whereas nNOS was observed only in perivascular nerves. sGC and PDE5 were expressed in smooth muscle. Thus, NO-sGC-PDE5 pathway is evolutionarily present in Crotalus sp. vessels, and has a remarkable degree of functional similarity to mammalian vessels.

Characterization of the urinary bladder dysfunction in renovascular hypertensive rats.

AIMS: Association between arterial hypertension and urinary bladder dysfunction has been reported in humans and spontaneously hypertensive rats. However, no study exists evaluating the bladder dysfunction in conditions of renovascular hypertension. The purpose of this study was to characterize the bladder dysfunction in two kidney-one clip (2K-1C) hypertensive rats. METHODS: A silver clip was placed around the renal artery of male Wistar rats. After 8 weeks, cystometric study, concentration-response curves to contractile and relaxant agents, frequency-dependent contractions, histomorphometry, muscarinic M(2) /M(3) mRNA expression and cyclic AMP measurements were performed. RESULTS: 2K-1C rats showed enhanced bladder volume, wall thickness and smooth muscle density. 2K-1C rats also exhibited increases in bladder capacity and non-void contractions, and decreases in the inter-contraction intervals. In isolated detrusor smooth muscle (DSM), contractions to carbachol and electrical-field stimulation (EFS) were significantly greater in 2K-1C rats. The Rho-kinase inhibitor Y27632 (10 µM) significantly reduced the carbachol-induced contractions in SHAM and 2K-1C rats, but DSM remained overactive in 2K-1C rats in presence of Y27632. Concentration-dependent contractions to the P2X receptor agonist α,ß-methylene ATP, KCl and extracellular Ca(2+) did not change between SHAM and 2K-1C groups. In 2K-1C rats, isoproterenol, metaproterenol and BRL 37-344 (non-selective, ß(2) - and ß(3) -selective adrenoceptor agonists, respectively) produced significantly lower relaxations and decreased cAMP levels, whereas relaxant responses to sodium nitroprusside and BAY 41-2272 remained unchanged. Muscarinic M(3) mRNA expression receptors were higher in 2K-1C group. CONCLUSIONS: Renovascular hypertensive rats exhibit bladder dysfunction that involves tissue remodeling and enhanced muscarinic M(3) -mediated contractions associated with reduced ß-adrenoceptor-mediated signal transduction.

The cholinergic response is increased in isolated ileum from gastroschisis rat model.

INTRODUCTION: Babies with gastroschisis (G) have high morbidity rate and long hospital stay due to bowel hypomotility caused by chronic exposure of the bowel to the amniotic fluid. Our aim was to evaluate the reactivity of isolated ileum in fetal rats selected for experimental gastroschisis. METHOD: G was surgically created at 18.5 days of gestation (term = 22 days). Concentration-dependent curve to the muscarinic agonist methacholine (1-30 µM) and contractions induced by electrical field stimulation (EFS, 1-16 Hz, 50 V, 1 ms) were carried out in isolated ileum of groups control (C), sham (S) and gastroschisis (G) (n = 30). Protein expression for M(3) was assessed by western blot analysis. RESULTS: The frequency and amplitude of spontaneous contractions were decreased in G (p < 0.001). Methacholine produced concentration-dependent contractions being the maximal response values higher in G (p < 0.01). EFS-induced frequency-dependent contractions showed 1.8 times higher in G as well as an increase of M(3) expression. CONCLUSION: The frequency and the amplitude of rhythmic contractions were reduced along with an increase in the contraction induced by mucarinic agonist and by EFS in G. These results suggest the occurrence of an adaptative supersensitivity to cholinergic response via increases in the protein expression for M(3) receptor.

Long-term oral treatment with BAY 41-2272 ameliorates impaired corpus cavernosum relaxations in a nitric oxide-deficient rat model.

OBJECTIVE: • To investigate the potential beneficial effects of 4-week oral treatment with 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1Hpyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272), a nitric oxide (NO)-independent soluble guanylate cyclase activator, on impaired rat corpus cavernosum relaxations in NO-deficient rats. MATERIAL AND METHODS: • Male Wistar rats were divided into four groups: Control, N (G)-nitro-L- arginine methyl ester (L-NAME; 20 mg/rat/day), BAY 41-2272 (20 mg/kg/day) and L-NAME + BAY 41-2272. • Rats were treated with L-NAME concomitantly with BAY 41-2272 for 4 weeks. • Concentration-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP), along with the nitrergic relaxations (1-32 Hz) were obtained in rat corpus cavernosum (RaCC). • The RaCC contractile responses to the α1 -adrenoceptor agonist phenylephrine (PE) were obtained. RESULTS: • Acetylcholine (0.01-1000 µmol/L) produced concentration-dependent relaxing responses in RaCC that were significantly enhanced (P < 0.05) in BAY 41-2272-treated rats. • The ACh-induced relaxations were largely reduced in L-NAME-treated rats, and co-treatment with BAY 41-2272 failed to significantly modify these impaired relaxations. • The SNP-induced relaxations were modified neither by L-NAME nor by co-treatment with BAY 41-2272. • The nitrergic relaxations were significantly amplified in BAY 41-2272-treated rats (at 16 and 32 Hz). A significant reduction in the nitrergic relaxations was observed in L-NAME-treated rats, an effect largely restored by co-treatment with BAY 41-2272. • The contractile RaCC responses produced by PE (0.001-100 µmol/L) were significantly higher (P < 0.05) in L-NAME-treated rats, and co-treatment of L-NAME with BAY 41-2272 nearly restored these enhanced contractile responses. CONCLUSION: • Four-week therapy with BAY 41-2272 prevents the impaired corpus cavernosum relaxations of rats treated chronically with L-NAME, indicating that accumulation of cyclic guanosine monophosphate into erectile tissue counteracts the NO deficiency.